Methods of expressing integrin β6 subunits

ABSTRACT

The present invention provides substantially pure integrins containing a novel β subunit designated as β 6 . The novel β 6  subunit forms heterodimers with α v  and α f . Methods of controlling cell adhesion using the β 6 -containing integrins are also provided.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a continuation of U.S. application Ser. No.10/219,631, filed Aug. 14, 2002, now U.S. Pat. No. 6,787,322, issuedSep. 7, 2004, which is a continuation of U.S. application Ser. No.10/072,841, filed Feb. 6, 2002, now U.S. Pat. No. 6,639,056, issued Oct.28, 2003, which is a continuation of U.S. application Ser. No.09/591,543, filed Jun. 8, 2000, now abandoned, which is a divisional ofU.S. application Ser. No. 08,938,085, filed Sep. 26, 1997, now U.S. Pat.No. 6,339,148, issued Jan. 15, 2002, which is a divisional of U.S.application Ser. No. 07/728,215, filed Jul. 11, 1991, now U.S. Pat. No.5,962,643, issued Oct. 5, 1999.

This work was supported in part by research grants HL/AL 33259, CA-47541and-CA-47858 from the National Institutes of Health. The U.S. Governmenthas rights in the invention.

BACKGROUND ART

This invention relates to receptors for adhesion peptides, and morespecifically to a novel receptor subunit having affinity forextracellular matrix molecules.

Multicellular organisms, such as man, have some 10¹⁴ cells which can bedivided into a minimum of fifty different types, such as blood cells andnerve cells. During the course of growth and development, cells adhereto other cells, or to extracellular materials, in specific and orderlyways. Such cell adhesion mechanisms appear to be of importance inmediating patterns of cellular growth, migration and differentiation,whereby cells develop specialized characteristics so as to function as,for example, muscle cells or liver cells. Cell adhesion mechanisms arealso implicated in dedifferentiation and invasion, notably where cellslose their specialized forms and become metastasizing cancer cells.

The mechanisms underlying the interactions of cells with one another andwith extracellular matrices are not fully understood, but it is thoughtthat they are mediated by cell surface receptors which specificallyrecognize and bind to a cognate ligand on the surface of cells or in theextracellular matrix.

The adhesion of cells to extracellular matrices and their migration onthe matrices is mediated in many cases by the binding of a cell surfacereceptor to an Arg-Gly-Asp containing sequence in the matrix protein, asreviewed in Ruoslahti and Pierschbacher, Science 238:491-497 (1987). TheArg-Gly-Asp sequence is a cell attachment site at least in fibronectin,vitronectin, fibrinogen von Willibrand, thrombopondin, osteopontin, andpossibly various collagens, laminin and tenascin. Despite the similarityof their cell attachment sites, these proteins can be recognizedindividually by their interactions with specific receptors.

The integrins are a large family of cell surface glycoproteins thatmediate cell-to-cell and cell-to-matrix adhesion as described, forexample, in the Ruoslahti and Pierschbacher article cited above. Allknown members of this family of adhesion receptors are heterodimersconsisting of an α and a β subunit noncovalently bound to each other.When the integrin family was first identified, integrins were groupedinto three subfamilies based on the three β subunits that were initiallyrecognized (β₁, β₂ and β₃). Over the past few years, the primarystructures of three integrin β subunits from mammalian cells and onefrom Drosophila have been deduced from cDNA.

Each α subunit was thought to associate uniquely with a single βsubunit. Eleven distinct α subunits have thus far been described. As newintegrins have been identified, however, it has become clear that thisgrouping is not entirely satisfactory, since there are clearly more thanthree β subunits and since some α subunits can associate with more thanone β subunit as described, for example, in Sonnenberg et al., J. Biol.Chem. 265:14030-14038 (1988).

Because of the importance of integrins in mediating critical aspects ofboth normal and abnormal cell processes, a need exists to identify andcharacterize different integrins. The present invention satisfies thisneed and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention relates to a substantially purified β subunit ofan integrin cell surface receptor designated as β₆. The amino acidsequence of human β₆ (SEQ ID NO:27) is provided in FIG. 3.

The present invention also relates to amino acid fragments specific toβ₆ that have a variety of uses. The invention further relates to vectorshaving a gene encoding such fragments. Host cells containing suchvectors are also provided. The nucleic acids encoding β₆ as well asnucleic acids that specifically hybridize with the nucleic acidsencoding β₆ sequences are other aspects of the present invention.

In a further aspect, the present invention relates to a substantiallypurified integrin comprising β₆ bound to an α subunit, particularlyα_(v) or α_(F). Methods of blocking the attachment of the β₆-containingintegrins to its ligand and of detecting the binding of such integrinsto its ligand are also provided.

The present invention also relates to methods of increasing ordecreasing cell adhesion in cells expressing a β₆-containing integrin byoverexpressing the integrin or by binding the integrin with a ligand,such as vitronectin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the design of consensus PCR primers (SEQ ID NOS:1-5, 7 and8)(β₂ human nucleic acids=SEQ ID NOS:10, 14, 18 and 22; corresponding β₂human amino acids=SEQ ID NOS:50, 51, 54 and 55; βhuman nucleic acids=SEQID NOS:11, 15, 19 and 23; corresponding β₃ human amino acids=SEQ IDNOS:52, 53, 56 and 57; β₁ human nucleic acids=SEQ ID NOS:12, 16, 20 and24; corresponding β₁ human amino acids=SEQ ID NOS:50, 53, 58 and 59; β₁chicken nucleic acids=SEQ ID NOS:13, 17, 21 and 25; corresponding β₁chicken amino acids=SEQ ID NOS:50, 53, 60 and 59: β₆ guinea pig sequencefrom position 219=SEQ ID NO:6; corresponding β₆ guinea pig aminoacids=SEQ ID NO:61; β₆ guinea pig sequence from position 1325=SEQ IDNO:8; corresponding 136 guinea pig amino acids=SEQ ID NO:62).

FIG. 2 shows a map of sequencing strategy.

FIG. 3 shows the nucleotide sequence and amino acid translation forhuman (H) (SEQ ID NOS:26 and 27) and guinea pig (GP) (SEQ ID NOS:28 and29) β₆ .

FIG. 4 shows the alignment of human β₆ (SEQ ID NO:27) with, fourpreviously reported integrin β subunits (human β₁ =SEQ ID NO:30; humanβ₂=SEQ ID) NO:31; human β₃=SEQ ID NO:32; Drosophila β_(myo)=SEQ IDNO:33).

FIG. 5 shows the alignment of partial nucleotide and amino acidsequences from human (H) and guinea pig (GP) β₁ (human(β_(1H))=SEQ IDNOS:34 and 35; guinea pig (β_(1GP))=SEQ ID NOS:36 and 37, respectively),β₃ (human(β₃)=SEQ ID NOS:38 and 39; guinea pig (β_(6GP))=SEQ ID NOS:40and 41, respectively), and β₆ (human(β_(6H)=SEQ ID NOS:42 and 43; guineapig (β_(6GP))=SEQ ID NOS:44 and 45, respectively) for the region justdownstream from the B3F primer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a composition of matter relating to anovel, substantially purified integrin β subunit, referred to herein asβ₆. The amino acid sequence of 62 ₆ for human (SEQ ID NO:27) and forguinea pig (SEQ ID NO:29) are also provided and are shown in FIG. 3.

By “substantially purified” is meant substantially free of contaminantsnormally associated with a native or natural environment.

By “β₆” is meant a polypeptide having substantially the same amino acidsequence and binding functions of the polypeptides encoded by thesequences set forth in FIG. 3 for human (SEQ ID NO:26) and guinea pig(SEQ ID NO:28) β₆. Thus, modified amino acid sequences that do notsubstantially destroy the functions and retain the essential sequence ofβ₆ are included within the definition of β₆. Amino acid sequences, suchas the sequence for β₁ (SEQ ID NO:30), β₂ (SEQ ID NO:31) and β₃ (SEQ IDNO:32), having less than 50% homology with the sequence of 62 ₆, are notsubstantially the same sequence and, therefore, do not fall within thedefinition of 62 ₆. Given the amino acid sequences set forth herein,additions, deletions or substitutions can be made and tested todetermine their effect on the function of β₆. In addition, one skilledin the art would recognize that certain amino acids, such as theconserved cystines, for example, can be modified to alter a bindingfunction of β₆.

Amino acids are identified herein by the standard one-letterabbreviations, as follows:

Amino Acid Symbol Alanine A Asparagine N Aspartic acid D Arginine RCysteine C Glutamine Q Glutamic acid E Glycine G Histidine H IsoleucineI Leucine L Lysine K Methionine M Phenylalanine F Proline P Serine SThreonine T Tryptophan W Tyrosine Y Valine V

Based on its amino acid sequence, the β subunit of the present inventionis clearly different from β₁, β₂, β₃ and other β subunits that haverecently been discovered. For example, the 11-amino acidcarboxyl-terminal extension on β₆ distinguishes it from β₁, β₂, and β₃.The short cytoplasmic tails of β₁, β₂, and β₃ are thought to be sites ofinteraction with the cytoskeleton and regions for the transduction ofsignals initiated by interactions of the large extracellular domainswith ligands. These cytoplasmic tails may also be targets for regulationof integrin function. The distinctive 11-amino acid cytoplasmic tail ofβ₆ indicates that its regulation or pathways for signal transduction maybe different from those of β₁, β₂ and β₃.

In addition to β₁, β₂ and β₃. recent studies have suggested theexistence of as many as five other integrin β subunits. A β subunit witha molecular weight of approximately 210,000 (β₄) has been foundassociated with the integrin α subunit “α₆” in colon carcinoma cells andin a variety of other tumor cells of epithelial origin as described, forexample, in Kajiji et al., EMBO J., 8:673-680 (1989). On the basis ofits high molecular weight, 210,000 compared with the predicted size of106,000 of the subject novel protein, and on the basis of its clearlydifferent amino-terminal sequence, it is apparent that β₄ is not thesame as the subject polypeptide.

Another β subunit, originally called β_(x) was identified inepithelial-derived tumor cells in association with the integrin αsubunit α_(v) as described, for example, in Cheresh et al., Cell57:59-69 (1989). This β subunit, having a distinctive amino-terminalsequence, was recently renamed β₅. Based on recent studies of purifiedpreparations, β₅ clearly differs from the β subunit of the presentinvention. Because the β subunit described in the present report isdistinct from each of the five β subunits for which sequence informationis available, it has been designated as β₆.

The existence of two other integrin β subunits has been inferred fromthe identification of unique proteins after immunoprecipitation ofsurface-labeled cell lysates with antibodies to known α subunits. One ofthese novel proteins, called β_(s) was found in association with α_(v)in the human osteosarcoma cell line MG-63, in the fibroblast cell lineAF1523, and in human endothelial cells as described, for example, inFreed et al., EMBO J. 8:2955-2965 (1989). This subunit is also differentfrom B₆ since β_(s) is expressed in MG-63 cells while β₆ is notexpressed in these cells as shown in Table 1.

The other novel integrin B subunit identified by co-immunoprecipitationof known α subunits, β_(P), is a protein of about M_(r) 95,000 that isfound to be associated with α₄, an α subunit first found as part of thelymphocyte homing receptor VLA-4 as described, for example, in Holzmannet al., Cell 45:37-46 (1989). This subunit is also distinct from β₆since β_(P) is expressed in lymphocytes while β₆ is not expressed inlymphocytes as shown in Table 1.

TABLE 1 Distribution of β₆ Type Results Source Cell Lines: FG-2Pancreatic + Kajiji et al., EMBO J. 3: 673–80 (1989) Panc I Pancreatic −Dr. Metzgar, Duke U., N.C. Colo-396 Colon CA + Dr. L. Walker, Cytel, SanDiego, CA UCLA P3 Lung CA + Dr. L. Walker, Cytel, San Diego, CA HeLaCervical − ATCC #CCL-2 Jar Chorio CA + ATCC #HTB 36 HT 1080 Fibrosarcoma− ATCC #CCL 121 U 937 Monocytoid − ATCC #CRL 1593 M 21 Melanoma − Dr. R.Reisfeld, Scripps Clinic & Research Foundation, La Jolla, CA B 16Melanoma − Dr. R. Reisfeld Scripps Clinic & Research Foundation, LaJolla, CA MG 63 Osteosarcoma − ATCC #CRL 1427 Tissues: Cervix + AorticEndothelium − Leukocytes −

The invention also provides an integrin comprising β₆ bound to an αsubunit. β₆, consistent with recent findings of other β subunits, canassociate with a variety of α subunits to form a functional integrin. Inone embodiment, β₆ associates with α_(v). In another embodiment, β₆associates with another α subunit referred to herein as α_(F). The α_(v)β₆ integrin, as well as other integrins containing β₆, can bindmolecules, for example extracellular matrix molecules. Such moleculesare referred to herein as ligands. In a specific embodiment, certainβ₆-containing integrins can bind Arg-Gly-Asp-containing polypeptidessuch as vitronectin or fibronectin. The binding of β₆-containingintegrins to various ligands can be determined according to proceduresknown in the art and as described for example, in Ruoslahti andPierschbacher, Science 238:491-497 (1987).

The invention also provides an amino acid fragment specific to β₆. Sinceβ₆ is a novel molecule, it contains many fragments which are specificfor this β subunit. Fragments specific to β₆ contain sequences havingless than 50% homology with sequences of other known integrin β subunitfragments. These fragments are necessarily of sufficient length to bedistinguishable from known fragments and, therefore, are “specific forβ₆.” The amino acid sequence of such fragments can readily be determinedby referring to the figures which identify the β₆ amino acid sequences.These fragments also retain the binding function of the β₆ subunit andcan therefore be used, for example, as immunogens to prepare reagentsspecific for β₆ or as an indicator to detect the novel β₆-containingintegrin of the present invention. One skilled in the art would know ofother uses for such fragments.

The invention also provides a reagent having specificity for an aminoacid sequence specific for β₆. Since β₆ is a novel protein with at least50% amino acid differences over related β subunits, one skilled in theart could readily make reagents, such as antibodies, which arespecifically reactive with amino acid sequences specific for β₆ andthereby immunologically distinguish β₆ from other molecules. Variousmethods of making such antibodies are well established and aredescribed, for example, in Antibodies, A Laboratory Manual, E. Harlowand D. Lane, Cold Spring Harbor Laboratory 1988, pp. 139-283 and Huse etal., Science 24:1275-1280 (1988).

The invention also provides nucleic acids which encode β₆. Examples ofsuch sequences are set forth in FIG. 3 (SEQ ID NOS:26 and 28). Followingstandard methods as described, for example, in Maniatis et al.,Molecular Cloning, Cold Spring Harbor (1982), nucleic acid sequences canbe cloned into the appropriate expression vector. The vector can then beinserted into a host, which will then be capable of expressingrecombinant proteins. Thus, the invention also relates to vectorscontaining nucleic acids encoding such sequences and to hosts containingthese vectors.

The sequences set forth in FIG. 3 (SEQ IS NOS:26 and 28) also providenucleic acids that can be used as probes for diagnostic purposes. Suchnucleic acids can hybridize with a nucleic acid having a nucleotidesequence specific for β₆ but do not hybridize with nucleic acidsencoding non-β₆ proteins, particularly other cell surface receptors.These nucleic acids can readily be determined from the sequence of β₆and synthesized using a standard nucleic acid synthesizer. Nucleic acidsare also provided which specifically hybridize to either the coding ornon-coding DNA of β₆.

Integrin cell surface receptors bind ligands, such as extracellularmatrix molecules. However, the binding of the integrin to the ligand canbe blocked by various means. For example, the binding of a β₆-containingintegrin can be blocked by a reagent that binds the β₆ subunit or theβ₆-containing integrin. Examples of such reagents include, for example,Arg-Gly-Asp-containing peptides and polypeptides, ligand fragmentscontaining the integrin binding site, as well as antibodies specificallyreactive with β₆ or a β₆-containing integrin. Alternatively, theblocking can be carried out by binding the ligand or fragment thereof,recognized by a β₆-containing integrin with a reagent specific for theligand at a site that inhibits the ligand from binding with theintegrin. Since the binding of a β₆-containing integrin to its ligandcan mediate cell adhesion to an extracellular matrix molecule,preventing this binding can prevent cell adhesion. Alternatively, celladhesion can be promoted by increasing the expression of β₆-containingintegrins by a cell.

Finally, the invention provides a method of detecting ligands which binda β₆-containing integrin. The method comprises contacting aβ₆-containing integrin with a solution containing ligands suspected ofbinding β₆-containing integrins. The presence of ligands which bind aβ₆-containing integrin is then detected.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE I Identification of a Novel β Subunit

A. Generation of cDNA Fragments by Polymerase Chain Reaction

Tracheal epithelial cells, harvested from male Hartley outbred guineapigs (Charles River Breeding Laboratories, Bar Harbor, Me.) were grownto confluence over 10-14 days on collagen-impregnated microporousfilters commercially available from Costar. RNA was harvested from theseprimary cultures, and mRNA was purified over oligo(dT)-cellulose columnsusing the Fast Track mRNA isolation kit (Invitrogen, San Diego, Calif.).Two to 5 μg of mRNA was used as a template for cDNA synthesis catalyzedby 200 units of Moloney murine leukemia virus reverse transcriptase(Bethesda Research Laboratories, Gaithersburg, Md.) in a 20-40 μlreaction volume. One to 5 μl of the resultant cDNA was used as atemplate for polymerase chain reaction (PCR). PCR was carried out in areaction volume of 25-200 μl. In addition to the template cDNA, each PCRreaction contained 50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25° C.), 1.5 mMMgCl₂, 0.01% gelatin, 0.1% Triton X-100, 0.2 mM each of dATP, dGTP, dCTPand dTTP, and 0.05 units/μl Taq DNA polymerase (obtained from eitherUnited States Biochemical Corporation, Cleveland, Ohio, or from Promega,Madison, Wis.).

For each reaction, two oligonucleotide primers were also added to obtaina final concentration of 1 μM each. The primer pairs are identifiedbelow. Each reaction mixture was overlaid with mineral oil, heated to950° C. for 4 min. in a thermal cycler (Ericomp, San Diego, Calif.), andthen subjected to 30 cycles of PCR. Each cycle consisted of 45 secondsat 95° C., 45 seconds at 53° C., and 1 min. at 72° C. Immediately afterthe last cycle, the sample was maintained at 72° C. for 10 min.

The results of each PCR reaction were analyzed by gel electrophoresis in1.5% agarose. Reactions that produced fragments of the expected sizewere electrophoresed in 1.5% low gel temperature agarose (Bio-RadLaboratories, Richmond, Calif.). The appropriate size band was excised,melted at 68° C., and the DNA was purified by extraction withphenol/chloroform and precipitation in ethanol and ammonium acetate.

B. PCR Primers

To obtain the initial fragment of the novel β subunit cDNA describedherein, degenerate mixtures of PCR primers were used. Oligonucleotideswere synthesized, trityl-on, by the University of California, SanFrancisco Biomolecular Resource Center using a DNA synthesizer withstandard procedures, and purified over Nen-sorb cartridges (DuPont-NewEngland Nuclear, Boston, Mass.). These consensus primer mixtures weredesigned to anneal with the nucleotides encoding the highly conservedsequence Asp-Leu-Tyr-Tyr-Leu-Met-Asp-Leu (SEQ ID NO:50) (primer B1F)(SEQ ID NO:1) and Glu-Gly-Gly-Phe-Asp-Ala-Ile-Met-Gln (SEQ ID NO:53)(primer B2R) (SEQ ID NO:2) that flank an approximately 300-nucleotideregion beginning approximately 130 amino acids from the amino terminusof each of the integrin β subunits sequenced to date. The sequences ofthe primers identified herein are depicted in FIG. 1 (SEQ ID NOS:1-8).

On the basis of the initial sequence obtained, a specific forward primerwas designed to anneal with the sequence encoding the amino acidsPro-Leu-Thr-Asn-Asp-Ala-Glu-Arg (SEQ ID NO:61) (primer BTE2F) (SEQ IDNO:7) ending approximately 49 nucleitides from the 3′ end of the regionthat had been sequenced. An additional forward primer (B3F) (SEQ IDNO:3) and two reverse primers (B3R and B4R) (SEQ ID NOS:4 and 5) werealso designed to recognize highly conserved consensus regions encodingthe sequences Gly-Glu-Cys-Val-Cys-Gly-Gin-Cys (SEQ ID NO:58) (B3 region)(SEQ ID NOS:3 and 4) and Ile-Gly-Leu-Ala-Leu-Leu-Leu-Trp-Lys (SEQ IDNO:59) (B3 region) (SEQ ID NO:5). The alignment of these primers withpreviously published sequences of human β₁, β₂, and β₃ and chicken β₁ isshown in FIG. 1. PCR as described above was performed with cDNA fromguinea pig trachel epithelial cells and the primer pairs BTE2F/B3R (SEQID NOS:7 and 4) and B3F/B4R (SEQ ID NOS:3 and 5).

The primer pair BTE2F/B3R (SEQ ID NOS:7 and 4) yielded 1095 additionalbase pairs of new sequence. Based on this sequence another specificprimer (BTE3F) (SEQ ID NO:8) was designed to recognize the sequenceVal-Ser-Glu-Asp-Gly-Val (SEQ ID NO:9) near the 3′ end of this sequence,and PCR was performed with this primer in combination with primer B4R(SEQ ID NO:5).

FIG. 1 shows the design of the PCR primers. β subunit consensus primermixtures were designed on the basis of alignment of published sequencesof human β₁, β₂, β₃ and chicken β₁. For forward primers (B1F and B3F)(SEQ ID NOS:1 and 3), the primer sequences included a single nucleotidewhenever possible for each of the first two nucleotides of each codonand were usually either degenerate or included deoxyinosine for thethird base in codons for amino acids other than methionine. Reverseprimers (B2R, B3R, and B4R) (SEQ ID NOS:2, 4 and 5) were designed in thesame manner for the complementary DNA strand. Two specific forwardprimers were designed to recognize β₆. The first (BTE2F) (SEQ ID NO:7)was designed to work across species and was thus degenerate or includeddeoxyinosine in the third codon position. The second, BTE3F (SEQ IDNO:8), was not degenerate and was designed to only recognize guinea pigβ₆.

C. Cloning of Fragments Obtained by PCR

Individual fragments were cloned in pBluescript (Stratagene, San Diego,Calif.) as follows. Purified fragments were resuspended in distilledwater containing deoxynucleotides and treated with 2.5 units of DNApolymerase I, large fragment (Promega) to fill in any 3′ recessed endsleft after the last cycle of PCR. The 5′ ends were phosphorylated with 5units of T4 polynucleotide kinase (New England Biolabs, Beverly, Mass.).An aliquot of the above reaction mixture containing approximately100-200 ng of DNA, was ligated into pBluescript that had been cut withEcoRV (Promega) and dephosphorylated with calf intestinal alkalinephosphatase (Boehringer Mannheim, Indianapolis, Ind.). Ligations wereperformed at 22° C. for 1 hour with T4 DNA ligase (Bethesda ResearchLaboratories). The ligation mixture was used to transform competentEscherichia coli (JM109, Clontech, San Francisco, Calif.). Plasmidscontaining inserts were purified using the Pharmacia miniprep lysis kit(Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.) denatured in 0.3 HNaOH, further purified over spin columns containing Sephacryl S-400(Pharmacia), and then sequenced using the Sequenase™ version 2.0sequencing kit (United States Biochemical Corp., Cleveland, Ohio) and[³⁵S]dATP (Amersham Corp., Arlington Heights, Ill.).

D. Library Screening

PCR fragments generated with the primer pairs B 1 F/B2R (SEQ ID NOS:1and 2) and BTE3F/B4R (SEQ ID NOS:8 and 5) were uniformly labeled withalpha-[³²P]dCTP and used as probes to screen a random-primed cDNAlibrary and an oligo-dT-primed cDNA library. Both libraries wereconstructed in the plasmid pTZl 8R-BstXI obtained from Invitrogen (SanDiego, Calif.) from mRNA obtained from the human pancreatic carcinomacell line FG-2. Plasmid was purified from clones found to hybridize witheither region and inserts were sequenced. A portion of insert DNA fromone clone was in turn labeled and used to screen the same libraries.Fourteen independent overlapping clones were sequenced from both endsusing primers that recognize regions of the pTZ polylinker. The regionsflanking the 3′ end of the putative translated region of the new βsubunit were sequenced in both directions from three clones usingprimers constructed to recognize sequences close to the 3′ end. On thebasis of the initial sequences thus obtained, an additional internalsequence was obtained from clones T10, T11, T12 and T14 (FIG. 2) afterdigestion with specific restriction endonucleases and religation. Threeinternal fragments thus generated were subcloned into pBluescript andwere also sequenced in both directions. Approximately 90% of the newsequence reported was obtained from both strands of DNA, and 97% wasobtained from two or more overlapping clones (FIG. 2).

FIG. 2 shows a map of the sequencing strategy. Shown are the location ofclones used to obtain the partial cDNA sequence of guinea pig β₆ (clones1F, 3L, 3N and 3Y, top) and the complete sequence of human β₆ (clonesT1-T19 bottom). Also shown is the location of the translated region(Protein). The location of the transmembrane domain is shown by theletters TM. Clones shown often represent one of several identicalclones. Internal sequence of clones with long inserts was obtained byrestriction endonuclease digestion and relegation and by ligation ofinternal fragments into pBluescript. Specific restriction sites employedare shown (Hind, HindIII; Hinc, HincII; Kpn, KpnI; Pst, PstI). Thedirection and extent of sequencing are shown by arrows. 1109 and 1110are the sites recognized by oligonucleotide sequencing primers. T18 andT19 each terminated in a poly(A) tail. The regions recognized by thedegenerate PCR primers B1F (B1), B2R (B2), B3R/F (B3), and B4R (B4) andthe β₆ primers BTE2F (BTE2) and BTE3F (BTE3) are noted above the guineapig cDNA map, kb, kilobases.

E. Nucleotide Sequence of a Novel Guinea Pig Integrin β Subunit

PCR using cDNA from guinea pig airway epithelial cells and the consensusprimer mixtures B1F and B2R (FIG. 1) amplified DNA fragments with theexpected size of approximately 350 nucleotides. When the fragment DNAwas sequenced after cloning into pBluescript, recombinant clones eachcontained inserts with one of two distinct sequences. One sequenceencoded a stretch of 98 amino acids that was 97% identical to theexpected region of human β₁ and was therefore presumed to be guinea pigB₁. The other sequence encoded 98 amino acids that were only 53%identical to human β₁, 45% identical to human β₂, and 57% identical tohuman β₃ (FIG. 2, clone 1F). Both of the guinea pig sequences includedthe integrin β subunit consensus sequences Ser-X-Ser-Met-X-Asp-Asp-Leu(SEQ ID NO:46) and Gly-Phe-Gly-Ser-Phe-Val (SEQ ID NO:47), and bothcontained the 2 cysteine residues found in this region in all knownintegrin β subunits. These data suggest that one of the two sequences weobtained encoded a new member of the integrin β subunit family.

This novel sequence was extended by further PCR steps utilizing primersspecific for the novel sequence (BTE2F, BTE3F) (SEQ ID NOS:7 and 8) incombination with two additional degenerate primers (B3R and B4R, seeFIGS. 1, 2 and 4). With the primer pair BTE2F/B3R (SEQ ID NOS7 and 4)two different cDNA products were obtained (3L and 3N in FIG. 2) due toan unexpected hybridization of the B3R primer with a site 220nucleotides further downstream (B3′ in FIG. 2). The 1732-nucleotidesequence determined from these clones is shown in FIG. 3.

FIG. 3 shows the nucleotide sequences and corresponding amino acidsequences for human (H) β₆ (SEQ ID NOS:26 and 27) and guinea pig (GP) β₆(SEQ ID NOS:28 and 29). The amino acid translation is denoted by thesingle letter code beneath the second nucleotide of each codon from thetranslated region of human β₆. For the guinea pig sequence, only aminoacids that differ from the human sequence are shown. The numbers alongthe right-hand margin denote the nucleotide or amino acid number of thelast entry on each line. The numbering system used starts with the firstnucleotide or amino acid available for each sequence shown. The ninepotential sites for N-glycosylation in the putative extracellular domainof human β₆ are underlined.

F. Nucleotide Sequence of Human B₆

Screening of cDNA libraries constructed from the human pancreaticcarcinoma cell line FG-2 with guinea pig cDNA probes 1F and 3Y (see FIG.2) and subsequent screening with a probe constructed from a portion ofclone T10 (FIG. 2) produced 14 independent positive clones. The twolongest clones (T18 and T19) extended to the poly(A) tail. A map ofthese clones, constructed on the basis of sequence information and ofthe mobility of inserts cut out of these clones in agarose gels is shownin FIG. 2. This map-predicts an mRNA of approximately 5 kilobasesincluding at least a 226-nucleotide untranslated region at the 5′ endand, a 2364-nucleotide open reading frame, and a 3′ untranslated regionof approximately 2.5 kilobases. This molecule has been termed integrinβ₆.

FIG. 3 shows the partial nucleotide and complete amino acid sequencesfor human β₆ (SEQ ID NOS:26 and 27) (excluding most of the3′-untranslated region) and the alignment of the 1732 nucleotides ofsequence obtained from PCR of guinea pig airway epithelial cell cDNA. Ofthe 577 amino acids deduced from the region sequenced in both speciesonly 36 residues differ; The amino acid sequences are 94% identical.Furthermore, of the 1732 nucleotides sequenced in both species, 91% areidentical. Nine potential glycosylation sites present in the putativeextracellular domain of human β₆ are shown by underlining. All seven ofthese sites that lie within the 577 amino acids obtained for guinea pigβ₆ are also present in the guinea pig protein.If all of the potentialglycosylation sites are occupied with oligosaccharides having an averagemolecular weight of 2,500, the predicted molecular weight of human β₆would be 106,000.

Comparison of the 788-amino acid sequence deduced from the open readingframe to the three previously sequenced human β subunits (SEQ IDNOS:30-32) and the myospheroid protein of Drosophila (SEQ ID NO:33) isshown in FIG. 4.

FIG. 4 shows the alignment of β₆ with four previously reported integrinβ subunits. Previously published sequences for human β₁ (SEQ ID NO:30),human β₂ (SEQ ID NO:31), human β₃ (SEQ ID NO:32), the myospheroid geneproduct (βmyo) of Drosophila (SEQ ID NO:33), and the novel sequencedescribed as β₆ (SEQ ID NO:27) are shown using the single letter aminoacid code. The 56 conserved cysteines are noted by * and the 120 otherinvariant amino acids by=above each line. The transmembrane domain isunderlined. The regions used for constructing the consensus β subunitprimers B1F (1) (SEQ ID NO:2), B2R (B2) (SEQ ID NO:2), B3F/R (B3) (SEQID NOS:3 and 4), and B4R (B4) (SEQ ID NO:5) are labeled below thealignment in bold type. The numbers along the right-hand margin denotethe number of the last amino acid in each line beginning from the firstamino acid of each putative signal sequence.

There are 179 amino acid residues that are identical, in each of theother β subunits and in β₆ including 56 conserved cysteine residues. Theoverall percentage of identical amino acids between β₆ and the otherhuman β subunits is 47% for β₃, 42% for β₁ and 38% for β₂. Human β₆ isalso 39% identical to the Drosophila β subunit. Human β₁, β₂ and β₃ andthe Drosophila β subunit all have cytoplasmic regions consisting of 41amino acids (beginning after the putative transmembrane domain shown bythe underline in FIG. 4). Although β₆ contains each of the 10 conservedamino acid residues in this cytoplasmic region it also contains an11-amino acid extension at the carboxyl terminus. β₆ also contains twoArg-Gly-Asp sequences, one at amino acids 514-516 and the other at594-596. These regions could serve as recognition sites for otherligands of the integrin family.

PCR using the primer pair B3F/B4R (SEQ ID NOS:3 and 5) (see FIG. 1)amplified fragments of the expected size of approximately 750nucleotides. Cloning and sequencing of the fragments did not result inany additional clones containing the novel β subunit sequence but didresult in several clones with inserts encoding an amino acid sequencethat was 97% identical to the corresponding region of human β₃ andseveral others encoding an amino acid sequence that was 93% identical tohuman β₁ (SEQ ID NO:35) (FIG. 5). These are presumably the guinea pighomologues of β₁ and β₃, respectively (SEQ ID NOS:37 and 41). Thenucleotide sequences of guinea pig (SEQ ID NO:36) and human β₁ (SEQ IDNO:34) are 80% identical, and those of guinea pig (SEQ ID NO:40) andhuman β₃ (SEQ ID NO:38) are 91% identical.

FIG. 5 shows the alignment of partial nucleotide and amino acidsequences from human (H) and guinea pig (GP) β₁ (SEQ ID NOS:34-37), β₃(SEQ ID NOS:38-41), and β₆ (SEQ ID NOS:42-45) for the region justdownstream from the B3F primer. Amino acid translations denoted by theone-letter code are shown below the second nucleotide of each codon. Forthe guinea pig sequences, only amino acids that differ from the humansequences are shown. The numbers shown along the right-hand margindenote the nucleotide number for human β₆. The sequences for human β₁and β₃ are from previously published reports.

EXAMPLE II β₆ Associates with α_(v) And α_(F) Subunits

To determine that the novel β subunit of the present invention isassociated with an α chain similar to other known integrins, antiseraagainst peptides from the cytoplasmic domain sequence of β₆ wereprepared. The following amino acid peptides from the cytoplasmicsequence of β₆ were prepared and used to immunize rabbits:RGSTSTFKNVTYKHR (SEQ ID NO:48) (residues 763-777) and YKHREKQKVDLSTDC(SEQ ID NO:49) (residues 774-788). The antisera were raised in rabbitsaccording to standard procedures known in the art. Briefly, peptideswere chemically coupled to keyhole limpet hemocyanin, and were injectedin rabbits in either complete (first injection only) or incompleteFreund's adjuvant as described, for example, in Antibodies: A LaboratoryManual, E. Harlow and D. Lowe, eds., Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. 11724. Antisera were termed 6830 (to peptidescorresponding to residues 763-777) and 6341 (to peptides correspondingto residues 774-788).

The resulting polyclonal antibodies were used to immunoprecipitatedetergent lysates from the pancreatic carcinoma cell line FG-2 that hadbeen surface radioiodinated according to procedures well known in theart such as described, for example, in Kajiji et al., EMBO J. 3:673-680(1989). A complex of two bands was precipitated of respectively 150kilodaltons (Kd) and 97 Kd in SDS-PAGE under non-reducing conditions.Under reducing conditions, the two bands migrated as a diffused band,extending from 130 Kd to 116 Kd. These bands were specific sincepre-immune serum did not precipitate any of them and they were notpresent when the immunoprecipitation was carried out in the presence ofthe corresponding immunogenic peptide. Furthermore, the same complex oftwo bands was precipitated by both the 6830 and 6841 antibodies, whichwere raised against independent peptides from the cytoplasmic sequencededuced from β₆ cDNA clones.

To determine which of the two precipitated bands corresponds to β₆, aSDS-heat denaturated lysate from surface-radioiodinated FG-2 cells wasimmunoprecipitated with the 6841 antibody. Only the 97 Kd band wasdetectable (non-reducing conditions), identifying it as the β₆ band.Under reducing conditions, the apparent molecular weight of this bandincreased to 116 Kd suggesting the presence of many intra-chaindisulfide bonds, which is consistent with the primary structure of β₆and of other integrin B chains.

The other band, of 150 Kd or 130 Kd under non-reducing or reducingconditions, respectively, is likely to be an α subunit since itdissociates after SDS-heat denaturation of the lysate, indicating thatit is non-covalently associated with the B₆ polypeptide. Furthermore,similar to certain other integrin α chains, its molecular weightdecreases under reducing conditions by about 20 Kd (130 Kd versus 150 Kdunder non-reducing conditions) probably due to a disulfide linked smallpeptide that dissociates upon reduction.

To identify which α chain is associated with β₆, the αβ₆ integrincomplex was purified by immuno-affinity chromatography on a 6841-proteinA sepharose matrix according to procedures well known in the art such asdescribed, for example, in Kajiji et al., EMBO J. 3:673-680 (1989). Theeluted material was immunoprecipitated with antibodies specific for α₁,α₂, α₃, α₅, α₆ and α_(v), which are known to be expressed in FG-2 cells.Only the anti-α_(v) monoclonal antibody 142.19, obtained from Dr. DavidCheresh, The Scripps Research Institution, La Jolla, Calif., reactedwith the purified material, which indicates that the α_(v) is associatedwith β₆ in this pancreatic carcinoma cell line.

To confirm this data, immunodepletion experiments onsurface-radioiodinated FG-2 lysates were performed according to methodswell known in the art such as described in Kajiji et al., EMBO J.3:673-680 (1989). The cell lysate was depleted with the 6841 anti-β₆antibody or, in parallel, with a control antiserum, and thenimmunoprecipitated with the 142.19 anti-α_(v) antibody. A smaller amountof α_(v) was present in the immunoprecipitation on the β₆ depletedlysate and no 97 Kd β₆ band was visible. Instead, a smaller band ofabout 90 Kd was present. It is hypothesized that this smaller bandrepresents the β₅ chain also associated with α_(v) in these cells. Inthe control lysate depleted with normal rabbit serum, all three bands,150 Kd (α_(v)), 97 Kd (β₆) and 90 Kd (β₅) were present afterimmunoprecipitation with the anti-α_(v) 142.19 antibody.

Another immunodepletion was carried out using 142.19 antibody as thedepleting antibody, or in parallel a mouse monoclonal as a controlantibody. Immunoprecipitations of α_(v)-depleted lysate with anti-α_(v)142.19 antibodies did not. show the presence of any band, indicatingthat all α_(v)-containing integrins had been removed. However, the 6841anti-β₆ antibody still precipitated a complex of two bands, onecorresponding to β₆, the other with a molecular weight close to that ofα_(v). This α chain, however, must differ from α_(v) since it isunreactive with anti-α_(v) monoclonal antibodies and is referred toherein as α_(F). In the control depleted lysates, the 6841 anti-β₆antibody precipitates much stronger bands, consistent with thepossibility that, in FG-2 cells, two β₆ integrins exist, α_(v)β₆ andα_(F)β₆.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the claims.

1. A method of blocking binding of αvβ6 to a ligand, the method comprising, contacting a cell in vitro with an antibody that binds a β₆ subunit of αvβ6, wherein the β6 subunit comprises SEQ ID NO:27, and wherein the cell is selected from the group consisting of a pancreatic cancer cell, a colon cancer cell, a lung cancer cell, and a chorio cancer cell, and wherein the ligand is fibronectin.
 2. The method of claim 1, wherein the cell is a pancreatic cell.
 3. The method of claim 1, wherein the cell is a colon cancer cell.
 4. The method of claim 1, wherein the cell is a lung cancer cell.
 5. The method of claim 1, wherein the cell is a chorio cancer cell. 